The time-averaged three-dimensional structures of an ever-increasing number of proteins have been determined by x-ray crystallography and NMR spectroscopy; in contrast, although numerous experimental and theoretical techniques have indicated that proteins have a complex dynamical behavior over many amplitude and temporal scales, the nature, amplitudes and rates of the structural fluctuations about the average conformations of biological macromolecules remain largely uncharacterized. In particular, the essential contributions of intramolecular dynamics to the biological function of proteins has been delineated only in very few instances. Comprehensive experimental information on the internal motions of proteins is critical to the improvement of biophysical theories of protein properties; to the complete mechanistic interpretation of kinetic processes, such as enzyme catalysis, ligand recognition and protein folding; to the de novo synthesis of proteins; and to the rational design of novel protein ligands, including pharmaceutical agents. Ribonuclease HI (RNase H) is an endonuclease that hydrolyzes the RNA moiety in RNA-DNA hybrid molecules. The enzyme is widely distributed in prokaryotic and eukaryotic organisms, and retroviral reverse transcriptase contains a C-terminal RNase H domain. RNase H is required for reverse transcription of retroviral RNA and has been implicated in the control of the origin of genomic replication in Escherichia coli, consequently, biophysical studies of RNase H provide insights into the biological activity of a critical enzyme and have important therapeutic consequences. The proposed research will ascertain the determinants of stability and biological activity by comparing the structural, dynamical and enzymatic properties of RNase H derived from E. coli and the extremely thermophilic bacterium Thermus thermophilus. Because protein stability and activity are strongly temperature dependent, conserved structural and dynamical determinants of function in E. coli and T thermophilus RNase H are hypothesized to exhibit similar responses to temperature perturbations at the physiological temperatures appropriate for the microorganisms. To test this hypothesis and its corollaries, enzyme kinetics of E. coli and T. thermophilus RNase H will be measured as a function of temperature, and the microscopic conformational and dynamical changes of the mesophilic and thermophilic proteins in response to temperature changes and substrate binding will be characterized by using NMR spectroscopy. In addition, new NMR techniques for measurement of laboratory and rotating-frame spin relaxation rate constants will be developed for investigations of conformational dynamics of RNase H.